Kit for detection of bacterial species by means of dna analysis

ABSTRACT

The present invention relates to the detection and identification of different bacterial species, all of which cause zoonosis, based on DNA analysis. More specifically, the invention provides the primers, probes, genes and genic regions required to apply a method for the simultaneous detection of bacteria and bacterial groups belonging to the genera  Anaplasma, Ehrlichia, Borrelia. Bartonella, Coxiella, Rickettsia  and  Francisella  based on Multiple PCR analysis by RLB (Reverse Line Blotting), in addition to providing a kit to carry out said analysis.

This application is a U.S. national phase application under 35 U.S.C.§371 of International Patent Application No. PCT/ES2006/070082 filedJun. 15, 2006, which claims the benefit of priority to Spanish PatentApplication No. 200501481 filed Jun. 17, 2005 and U.S. ProvisionalPatent Application No. 60/691,231 filed Jun. 17, 2005, all of which arehereby incorporated by reference in their entireties. The InternationalApplication was published in Spanish on Dec. 28, 2006 as WO 2006/136639.

FIELD OF THE INVENTION

The present invention relates to the detection and identification ofdifferent bacterial species, all of which cause zoonosis, based on DNAanalysis. More specifically, the invention provides a method and kit forthe simultaneous detection of bacteria and bacterial groups belonging tothe genera Anaplasma, Ehrlichia, Borrelia, Bartonella, Coxiella,Rickettsia and Francisella based on DNA amplification.

BACKGROUND

To date, about 200 zoonotic diseases (bartonelosis, leptospirosis, Lymeborreliosis, etc.), which affect humans have been described. In thirdworld countries, they represent one of the main causes of death andentail substantial economic loss. Coexistence with animals, lack ofsanitary infrastructure and low cultural level continue to be the mainallies of these diseases.

Certain types of zoonosis are now thriving in industrialized countriesas a consequence of population increases in urban and periurban areas,and increased movement of animals across international borders, whichentails the risk of introducing exotic diseases into the environment.

These circumstances, coupled with the frequent findings of arthropodsinfected by more than one of the pathogens included in the presentinvention, increase the possibility of more than'one of the bacterialspecies included in the present invention being transmitted in a singlesting.

As a result, hospitalizations due to medical profiles produced by humancontact with animals or different classes of arthropods, such asmosquitoes, ticks, fleas, lice, mites, etc., which act as vectors orpathogen reservoirs, is becoming increasingly common. Said medicalprofiles, due to their high degree of similarity, do not allow a fastand reliable identification of the pathogenous agent, so that specificand fast treatment is not possible and is occasionally administered toolate. This undoubtedly justifies the need for a comprehensive detectionmethod.

The diagnostic methods currently available are limited to detectingantibodies which, in general, is retrospective and of little use totreating patients in acute-phase states. Culture is not considered adiagnostic method, due both to its technological complexity, whichexcludes it from regular practice in hospital microbiology laboratories,and to the need for P3 facilities.

Molecular diagnosis by genome amplification by means of PCR represents adiagnostic option of great value. However, clinical samples ofsufficient quantity for pathogen testing or the methodology required tocarry out different tests are not always available.

A paper has recently been published (Blaskovic D. et al. 2005.Oligo-based detection of tick-borne bacteria. FEMS Microbiology Letters243:273-8) which describes a method for the detection of 5 out of 6pathogens proposed by the present invention. Said method is based onribosomal DNA analysis and uses universal primers, which amplify thegenetic material of both target and non-target bacteria, due to whichits sensitivity is substantially reduced.

Other methods, such as those described by U.S. Pat. Nos. 6,300,072 and6,518,020, are capable of detecting and identifying bacteria of thegenus Bartonella, by using the same DNA region (intergenic space16S-23S). However, the number of species within this genus has increasedsubstantially since said patents were filed and their approximation,which consists of discriminating between species according to the sizeof the amplicon obtained during PCR, is not useful for certain knownspecies within the same genus which are similar in size to the amplifiedfragment.

While the method provided by the present invention also proposes usingintergenic region 16S-23S for the detection of species belonging to thegenus Bartonella, improvements have been introduced with respect to thepreviously described procedures, as it is capable of detecting a muchwider range of species within the same and other genera, usingcompletely new probes and primers with maximum sensitivity levels.

For the detection of Coxiella burnetii, the present invention uses thesame primers and DNA region (insertion sequence IS1111) as thepreviously described methods. Said detection has been improved bycombining it with another series of completely new tests aimed atidentifying other bacterial species, which can be transmitted by thesame vectors and also provide a new hybridization probe for thedetection of Coxiella burnetii.

DETAILED DESCRIPTION

Definitions: Multiple PCR or Multiplex PCR: PCR (Polymerase ChainReaction) is a system whereby the number of copies of a specificnucleotide sequence of an organism is amplified or increased using twoprimers. Multiple PCR or Multiplex PCR is a variation of PCR whichallows the simultaneous amplification of more than one target sequenceusing more than one pair of primers.

The present invention solves the problem of the tediousness andcomplexity of detecting a high number of bacteria that cause zoonosiswhich can be clinically and/or epidemiologically indistinguishable,through the development of a method and Kit for the simultaneousdetection of bacterial species that cause zoonosis belonging to thegenera: Anaplasma, Ehrlichia, Borrelia, Bartonella, Coxiella, Rickettsiaand Francisella.

The solution found by the present invention includes simultaneouslyanalyzing different bacterial DNA regions to determine which species arepresent in both cases. Specifically, the 16S rRNA gene is analyzed inorder to detect the presence of Anaplasma, Ehrlichia and Borrelia;intergenic space 23S-5S rRNA is analyzed to detect the presence ofRickettsia; the gene which codes for the precursor of the main membraneprotein TUL4 is analyzed to detect the presence of Francisella; thetransposase IS1111 gene is analyzed to detect the presence of Coxiellaand intergenic space 16S-23S is analyzed to detect the presence ofBartonella.

According to the above, a first aspect of the invention relates to amethod for the sample-based detection of bacteria, comprised of thefollowing steps:

-   -   i) Placing the sample under analysis in contact with a reaction        mixture containing specific primers to carry out Multiplex PCR.    -   ii) Amplifying by means of polymerase chain reaction.    -   iii) Identifying the formation of the products in the previous        step, said information being indicative of the presence or        absence of zoonosis-causing bacteria.

In relation to this first aspect of the invention, said inventionprovides a method to simultaneously detect:

-   -   Anaplasma phagocytophilum, A. bovis, A. equi, A. marginale, A.        centrale and A. ovis.    -   Ehrlichia chaffeensis and E. Ewingii.    -   Bartonella henselae, B. quintana, B. clarridgeiae, B.        elizabethae, B. grahamii, B. vinsonii subspecies berkhofii, B.        vinsonii subspecies vinsonii, B. vinsonii subspecies        aurupensis, B. bacilliformis, B. alsatica, B. bovis, B.        doshiae, B. koehlerae, B. schoenbuchensis, B. taylori and B.        tribocorum.    -   All of the species belonging to the genus Borrelia.    -   Coxiella burnetii.    -   Any subspecies of Francisella turalensis, including F.        tularensis subsp. tularensis, F. tularensis subsp. holarctica        and F. tularensis subsp. novicida which are jointly detected,        and variant 3523 of the same species and so-called endosymbionts        of different species of ixodides and argasides, which are        detected differentially.    -   The genus Rickettsia, and group that causes spotted fever and        the group that causes typhus, the species Rickettsia akari, R.        beffii, R. slovaca, R. conorii, R. aeschlimannii, R.        ricketsii, R. sibirica, R. helvetica, R. felis, R. australis, R.        prowazekii and R. typhy (R. mooserii).        all of which are capable of causing zoonosis, jointly infecting        an individual and being difficult to identify by simple        observation of medical profiles, based on the amplification and        analysis of the genes or specific genic regions presented in        Tables 1-6.

According to a specific embodiment of this first aspect of theinvention, DNA fragments included or comprised within the sequences, theaccess numbers of which are shown in Tables 1-6 below, are amplified.

According to a more specific embodiment of this first aspect of theinvention, the amplified regions have a size of between 99 and 686nucleotides and contain variable regions used for identification.According to an even more specific embodiment of the invention, thevariable regions contain or are included within sequences SEQ ID NO:55to SEQ ID NO:93 or complementary sequences, the positions of which areshown in Tables 1 to 6.

According to another embodiment of this first aspect of the invention,the amplification products that allow different bacterial species andgroups to be identified are detected by means of probes. According to amore preferred embodiment, said probes have a length of between 15 and25 nucleotides. And, according to an even more preferred embodiment, theprobes have sequences which comprise or are included within sequencesSEQ ID NO:3-6; SEQ ID NO:9-24; SEQ ID NO:27; SEQ ID NO:30; SEQ IDNO:33-35; SEQ ID NO:38-51; or complementary sequences (Tables 1-6).

The primers can be designed by means of multiple alignment usingcomputer programs such as CLUSTAL X, which allows the identification ofhighly conserved regions that act as moulds. According to anotherspecific embodiment of this first aspect of the invention, the primershybridize the genes indicated in Tables 1 to 6 below and particularlythose with sequences with the access numbers shown in Tables 1-6 below.According to an even more specific embodiment, the primers havesequences which comprise or are included within SEQ ID NO:1-2; SEQ IDNO:7-8; SEQ ID NO:25-26; SEQ ID NO:28-29; SEQ ID NO:31-32; SEQ IDNO:36-37; or complementary sequences.

Brief Explanation of the Tables:

-   -   Column 1 (organism) indicates the bacterial species or group of        bacterial species detected in each case.    -   Column 2 (gene) indicates the gene or genome region used to        detect the bacterial species or group of species in column 1.    -   Column 3 (primer) indicates the sequence of the pair of primers        required to carry out the amplification of variable gene regions        or genome regions indicated in each Table (column 2).    -   Column 4 (probe) indicates the sequence of the probes used to        detect the bacterial species or group of species referenced in        column 1 of each Table.    -   Column 5 (sequence 5′-3′) indicates the sequence references of        the variable regions which are amplified to detect each        bacterial species or group of species.    -   Column 6 (position 5′-3′):        -   The first row: indicates a sequence code relative to a gene            or genome region referenced in column 2, in addition to the            specific position of said sequence in which the primer is            hybridized (column 3).        -   The second to the last row of each Table indicate a sequence            code relative to a gene or genome region referenced in            column 2, in addition to the specific position of said            sequence to which the probe is joined (column 4).

TABLE 1 Anaplasma and Ehrlichia: sequence of each of the primers andprobes used in the process and their relative position within gene 16SrRNA. SEQUENCE 5′- POSITION 5′- ORGANISM GENE PRIMER PROBES 3′ 3′Anaplasma spp 16S SEQ ID 1  9-30 Ehrlichia spp (16S/AE-F) (U02521) SEQID 2 109-86  (16S/AE-R) (U02521) Anaplasma 16S SEQ ID 1 SEQ ID 3 SEQ ID57 52-73 phagocytophilum (16S/AE-F) (S-PHA) (U02521) A. bovis SEQ ID 2 8-29 A. equi (16S/AE-R) (AF470698)  8-29 (AF172167) Ehrlichiachaffeensis 16S SEQ ID 1 SEQ ID 4 SEQ ID 58 51-71 (16S/AE-F) (S-CHA)(AF147752) SEQ ID 2 (16S/AE-R) E. ewingii 16S SEQ ID 1 SEQ ID 5 SEQ ID59 46-66 (16S/AE-F) (S-EWI) (U96436) SEQ ID 2 (16S/AE-R) A. marginale16S SEQ ID 1 SEQ ID SEQ ID 60 53-71 A. centrale (16S/AE-F) NO 6 (S-(AJ633048) A. ovis SEQ ID 2 MCO) 72-90 (16S/AE-R) (AF414869) 72-90(AF414870)

TABLE 2 Bartonella: sequence of each of the primers and probes used inthe process and their relative position within intergenic space 16S-23SrRNA. SEQUENCE ORGANISM GENE PRIMER PROBES 5′-3′ POSITION 5′-3′Bartonella 16S- SEQ ID 7 494-515 spp. 23S (BAR/16-23F) (AF369527) SEQ ID8 908-889 (BAR/16-23R) (AF369527) B. henselae 16S- SEQ ID 7 SEQ ID 9 (S-SEQ ID 61 793-814 23S (BAR/16-23F) HENS) (AF369527) SEQ ID 8(BAR/16-23R) B. quintana 16S- SEQ ID 7 SEQ ID 10 (S- SEQ ID 62 622-64123S (BAR/16-23F) QUIN) (AF368396) SEQ ID 8 B. clarridgeiae 16S- SEQ ID 7SEQ ID 11 (S- SEQ ID 63 512-531 23S (BAR/16-23F) CLAR) (AF312497) SEQ ID8 (BAR/16-23R B. elizabethae 16S- SEQ ID 7 SEQ ID 12 (S- SEQ ID 64807-827 23S (BAR/16-23F) ELIZ) (L35103) SEQ ID 8 (BAR/16-23R B. grahamii16S- SEQ ID 7 SEQ ID 13 (S- SEQ ID 65 491-514 23S (BAR/16-23F) GRAH2)(AJ269790) SEQ ID 8 (BAR/16-23R B. vinsonii 16S- SEQ ID 7 SEQ ID 14 (S-SEQ ID 66 2242-2261 berkhofii 23S (BAR/16-23F) VIN-B) (AF143446) SEQ ID8 (BAR/16-23R B. vinsonii 16S- SEQ ID 7 SEQ ID 15 (S- SEQ ID 67 686-706arupensis 23S (BAR/16-23F) VIN-A1) (AF442952) SEQ ID 8 (BAR/16-23R B.vinsonii 16S- SEQ ID 7 SEQ ID 16 (S- SEQ ID 68 821-841 vinsonii 23S(BAR/16-23F) VIN-A2) (AF312504) SEQ ID 8 (BAR/16-23R B. bacilliformis16S- SEQ ID 7 SEQ ID 17 (S- SEQ ID 69 474-493 23S (BAR/16-23F) BACI)(AJ422181) SEQ ID 8 (BAR/16-23R B. alsatica 16S- SEQ ID 7 SEQ ID 18 (S-SEQ ID 70 589-608 23S (BAR/16-23F) ALS) (AF312506) SEQ ID 8 (BAR/16-23RB. bovis 16S- SEQ ID 7 SEQ ID 19 SEQ ID 71 455-478 23S (BAR/16-23F)(S-BOV2) (AY116638) SEQ ID 8 (BAR/16-23R B. doshiae 16S- SEQ ID 7 SEQ ID20 SEQ ID 72 724-743 23S (BAR/16-23F) (S-DOSH) (AJ269786) SEQ ID 8(BAR/16-23R B. koehlerae 16S- SEQ ID 7 SEQ ID 21 SEQ ID 73 778-803 23S(BAR/16-23F) (S-KOE) (AF312490) SEQ ID 8 (BAR/16-23R B. schoenbuchensis16S- SEQ ID 7 SEQ ID 22 SEQ ID 74 446-466 23S (BAR/16-23F) (S-SCHO2)(AY116639) SEQ ID 8 (BAR/16-23R B. taylori 16S- SEQ ID 7 SEQ ID 23 SEQID 75 655-673 23S (BAR/16-23F) (S-TAY) (AJ269784) SEQ ID 8 (BAR/16-23RB. tribocorum 16S- SEQ ID 7 SEQ ID 24 SEQ ID 76 692-713 23S (BAR/16-23F)(S-TRIB) (AF312505) SEQ ID 8 (BAR/16-23R

TABLE 3 Barrelia: sequence of each of the primers and probes used in theprocess and their relative position within gene 16S rRNA. SEQUENCE 5′-ORGANISM GENE PRIMER PROBES 3′ POSITION 5′-3′ Borrelia spp. 16S SEQ ID25 336-356 (BOF-3) (AJ224139) SEQ ID 26 567-547 (BOR) (AJ224139)Borrelia 16S SEQ ID 25 SEQ ID 27 (SG- SEQ ID 77 364-383 (BOF-3) BOR3)(AJ224139) SEQ ID 26 (BOR)

TABLE 4 Coxiella: sequence of each of the primers and probes used in theprocess and their relative position within insertion sequence(transposase) IS1111. ORGANISM GENE PRIMER PROBES SEQUENCE 5′-3′POSITION 5′-3′ Coxiella Trans- SEQ ID 28 200-211 burnetii posase (TRANS(M80806) IS1111 1) SEQ ID 29 885-865 (TRANS (M80806) 2) Coxiella Trans-SEQ ID 28 SEQ ID SEQ ID 78 520-539 burnetii posase (TRANS 30 (S-(M80806) IS1111 1) SEQ IS1111) ID 29 (TRANS 2)

TABLE 5 Francisella: Amplified gene, sequence of each of the primers andprobes used in the process and their relative position. POSITION 5′-ORGANISM GENE PRIMER PROBES SEQUENCE 5′-3′ 3′ Francisella 17 kDa SEQ ID31 593-617 spp. Tul4 (FT594) (M32059) SEQ ID 32 825-804 (FT827) (M32059)F. tularensis 17 kDa SEQ ID 31 SEQ ID SEQ ID 79 658-680 Tul4 (FT594) 33(S- (M32059) SEQ ID 32 TUL) FT827 Variant 17 kDa SEQ ID 31 SEQ ID SEQ ID80 169-188 3523 Tul4 (FT594) 34 (S- (AY243029) SEQ ID 32 TUL3523) FT827Endosymbionts 17 kDa SEQ ID 31 SEQ ID SEQ ID 81 533-553 Tul4 (FT594) 35(S- (AY375423) SEQ ID 32 ENDOS2) (FT827)

TABLE 6 Rickettsia: sequence of each of the primers and probes used inthe process and their relative position within genes 238, 58 rRNA andwithin intergenic space 23S-5S rRNA. SEQUENCE ORGANISM GENE PRIMERPROBES 5′-3′ POSITION 5′-3′ Rickettsia 23S-5S SEQ ID 36  1-22 spp.(RCK/23-5-F) (AY125012) SEQ ID 37 388-367 (RCK/23-5-R) (AY125012)Generic 23S-5S SEQ ID 36 SEQ ID SEQ ID 82 51-71 (RCK/23-5-F) 38 (SG-(AY125012) SEQ ID 37 RICK) (RCK/23-5-R) Spotted Fever 23S-5S SEQ ID 36SEQ ID SEQ ID 83 123-141 Group (RCK/23-5-F) 39 (SG- (AY125012) SEQ ID 37SFG) (RCK/23-5-R) R. akari 23S-5S SEQ ID 36 SEQ ID SEQ ID 84291.105-291.126 (RCK/23-5-F) 40 (S- (AAFE01000001) SEQ ID 37 AKA4)(RCK/23-5-R) R. bellii 23S-5S SEQ ID 36 SEQ ID SEQ ID 85 2721-2743(RCK/23-5-F) 41 (S- (U11015) SEQ ID 37 BELLII) (RCK/23-5-R) R. slovaca23S-5S SEQ ID 36 SEQ ID SEQ ID 86 194-211 (RCK/23-5-F) 42 (S- (AY125009)SEQ ID 37 SLO) (RCK/23-5-R) R. conorii 23S-5S SEQ ID 36 SEQ ID SEQ ID 87186-204 (RCK/23-5-F) 43 (S- (AY125012) SEQ ID 37 CON) (RCK/23-5-R) R.aeschlimannii 23S-5S SEQ ID 36 SEQ ID SEQ ID 88 183-204 (RCK/23-5-F) 44(S- (AY125016) SEQ ID 37 AESCH) (RCK/23-5-R) R. rickettsii 23S-5S SEQ ID36 SEQ ID SEQ ID 89 2814-2833 R. sibirica (RCK/23-5-F) 45 (S- (U11022)SEQ ID 37 RI/SI) (RCK/23-5-R) R. helvetica 23S-5S SEQ ID 36 SEQ ID SEQID 90 360-342 (RCK/23-5-F) 46 (S- (AY125017) SEQ ID 37 HELV)(RCK/23-5-R) R. felis 23S-5S SEQ ID 36 SEQ ID SEQ ID 55 186-207(RCK/23-5-F) 47 (S- (SEQ ID 55) SEQ ID 37 FEL) (RCK/23-5-R) R. australis23S-5S SEQ ID 36 SEQ ID 48 SEQ ID 56 230-249 (RCK/23-5-F) (S-AUS) SEQ ID37 (RCK/23-5-R) Grupo Tifus 23S-5S SEQ ID 36 SEQ ID 49 SEQ ID 912804-2827 (RCK/23-5-F) (SG-TG) (U11018) SEQ ID 37 (RCK/23-5-R) R.prowazekii 23S-5S SEQ ID 36 SEQ ID 50 SEQ ID 92 2824-2846 (RCK/23-5-F)(S-PROW) (U11018) SEQ ID 37 (RCK/23-5-R) R. typhi (R. mooserii) 23S-5SSEQ ID 36 SEQ ID 51 SEQ ID 93 188-211 (RCK/23-5-F) (S-TYPHI) (AY125019)SEQ ID 37 (RCK/23-5-R)

Given the abundance of PCR inhibitors, such as humic and fulvic acid,heavy metals, heparin, etc. which can produce false negatives and,despite the methods that exist to reduce the concentration of this typeof molecules, we recommend (cf. J. Hoorfar et al., “Making internalAmplification control mandatory for diagnostic PCR” J. of ClinicalMicrobiology, December 2003, pp. 5835) that the PCR tests contain anInternal Amplification Control (IAC). Said IAC is no more than a DNAfragment which is amplified simultaneously with the target sample, insuch a way that its absence at the end of the testing process indicatesthe presence of factors which have caused unwanted development of thePCR.

A second aspect of the invention relates to a method similar to thatdescribed in the first aspect of said invention, including at least oneIAC, preferably comprised of a DNA sequence of the TetrahydrocannabinolSynthase gene of the Cannabis sativa species and, more preferably, of asequence with access number AB183705.

According to a more preferred embodiment, a region of the AB183705sequence is amplified, said sequence being included within SEQ ID NO:94or complementary sequences (Table 7).

According to another preferred embodiment, the region is amplified bymeans of specific primers, the sequences of which comprise or areincluded within SEQ ID NO:52 and SEQ ID NO:53 or complementarysequences.

TABLE 7 Internal control: Amplified gene, sequence of each of theprimers and probes used in the process and their relative position.ORGANISM GENE PRIMER PROBES SEQUENCE 5′-3′ POSITION 5′-3′ IAC THC SEQ ID77-99 (Cannabis Synthase 52 (AB183705) sativa) (CI-F) SEQ ID 447-427 53(CI-R) (AB183705) Cannabis THC SEQ ID SEQ ID SEQ ID 94 281-302 sativaSynthase 52 54 (AB183705) (CI-F) (S-CI2) SEQ ID 53 (CI-R)

Within the context of this description, the term “specific” implies thatthe primers comprise a nucleotide sequence fully complementary to thegenes or genic fragments used by the present invention.

The term “variable regions” refers to DNA sequences which allow for theidentification of the bacterial species and groups identified by thepresent invention.

According to another embodiment of the second aspect of the invention,IAC amplification is detected by means of hybridization with probes.According to a more preferred embodiment, said probes have a length of15 to 25 nucleotides. And, in an even more preferred embodiment, saidprobes have a sequence comprised or included in SEQ ID NO:54 orcomplementary sequences.

The method provided by the present invention allows for the detection ofthe aforementioned bacteria and bacterial groups, independent of sampleorigin. Said samples may be obtained from biopsies, scrapings, insects,biological fluids (blood, urine, saliva, etc.), field, etc. Once taken,the sample is pretreated in order to carry out Multiple PCR andsubsequent amplicon identification.

The invention also provides diagnosis kits to apply the method describedby the invention, which contain:

-   -   Specific primers with sequences: SEQ ID 1-2, SEQ ID 7-8, SEQ ID        25-26, SEQ ID 28-29, SEQ ID 31-32, SEQ ID 36-37 and optionally        SEQ ID 52 y 53 as IAC.    -   Probes with sequences: SEQ ID 3-6, SEQ ID 9-24, SEQ ID 27, SEQ        ID 30, SEQ ID 33-35, SEQ ID 38-51 and optionally SEQ ID 54        (S-Cl2) as IAC.

In the same way, said kits can include all the reactive agents requiredto apply any of the methods described. This includes, without any typeof limitation, the use of buffers, polymerases, cofactors to optimizetheir activity, contamination-preventing agents, etc: On the other hand,the kits can include all of the supports and containers required fortheir startup and optimization.

The advantages of the present method and the kits with which to apply itinclude: speed (39 species and bacterial groups can be detected in lessthan 8 hours), specificity (the initiators used are specific to eachspecies or bacterial group) and a high level of sensitivity.

Within the context of the specification description and claims, the word“comprises” and its variations, such as “comprising”, does not intend toexclude other additives, components, constituent elements or stages.Both the examples and complementary drawings do not intend to limit theinvention, but should rather be considered an aid to better understandit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Hybridization membrane showing the validation of primers,probes, and variable regions for the detection of Anaplasma andEhrlichia (A), Borrelia (B), Francisella (C), Bartonella (D) andCoxiella (E) species, by means of specific probes (Tables 1-5). TheS-Cl2 probe refers to the IAC probe (Table 7).

FIG. 2. A) Hybridization membrane showing the validation of primers,probes and variable regions for the detection of Rickettsia species; B)Hybridization membrane showing an example of simultaneous detection ofspecies belonging to the 7 genera. In this example: A. phagocytophilum,A. marginate. E. chaffeensis, E. ewingi, B. henselae, B. burgdorferi, F.tularensis tularensis, R. conorii and R. prowazekii, tested at 10³, 10²and 10 equivalent genome/copies. In both cases (A and B) the S-Cl2probe, which refers to the IAC probe (Table 7), is used.

FIG. 3. Hybridization membrane showing the results of a specificitystudy carried out within the indicated group of probes (Tables 1-7) ondifferent species of bacteria, arthropods and mammals. The resultsreveal that the probes are not joined to the samples of the organismstested in any case. The S-Cl2 probe refers to the IAC probe (Table 7).

EXAMPLE

The present invention is next described by reference to an example. Theuse of this and other examples anywhere in the specification isillustrative only, and in no way limits the scope and meaning of theinvention or of any exemplified form. Likewise, the invention is notlimited to any particular preferred embodiments described herein.Indeed, modifications and variations of the invention may be apparent tothose skilled in the art upon reading this specification, and can bemade without departing from its spirit and scope. The invention istherefore to be limited only by the terms of the claims, along with thefull scope of equivalents to which the claims are entitled.

Alignments, Primer, and Probe Designs

Conserved regions were identified by comparing and aligning multiplesequences obtained from public databases such as Genbank(http://www.ncbi.nlm.nih.gov), based on which specific primers weredesigned (Tables 1-6) for use in Multiple PCR. The compatibility betweenthe primers, in addition to their optimal concentration, was empiricallytested in the same manner as the magnesium salts and bovine sericalbumin.

Variable regions were identified based on the selected sequencealignments, which allowed probes to be designed for the differentiationof bacterial species and genic groups (Tables 1-6) through RLB (ReverseLine Blotting) (Kaufhold A, Podbielski A, Baumgarten G, Blokpoel M, TopJ, Schouls L. Rapad Typing of group-a streptococci by the use of DNAamplification and nonradioactive allele-specific oligonucleotide probes.FEMS Microbiology Letters 119: 19-25 (1994)).

A first specificity analysis of each of the probes was carried out bycomparing its sequence against public databases (Genbank) using computerprograms such as BLAST (http://www.ncbi.hlm.nih.gov/blast/). Specificitywas subsequently demonstrated by carrying out tests on a variety of DNAsamples of different bacterial and eucaryotic species (FIG. 3).

DNA Culture Mediums and Isolation

The species and genic groups selected for identification were obtainedfrom private collections: the Spanish Type Culture Collection (CECT)and/or sample bank available at the National Microbiology Center (CNM).All of the species analyzed are shown in Table 8.

The isolation of genetic material was carried out using well-knownprocedures within the art and available on the market (DNA Mini Kit,Qiagen, N. Reference: 51304).

TABLE 8 Origin of DNA used in the invention NATIVE DNA SYNTHETICORGANISM (origin) DNA* Anaplasma phagocytophilum X (1) A. marginale X(1) Ehrlichia chaffeensis X E. ewingii X Borrelia burgdorferi X (2) B.garinii X (2) B. afzelii X (2) B. lusitaniae X (2) B. japonica X (2) B.hermsii X (2) B. parkeri X (2) Francisella tularensis tularensis X (3)F. tularensis subesp. holarctica X (3) F. tularensis subesp. Novicida X(3) Francisella variant 3523 X Francisella Endosimbiontes X Bartonellaalsatica X (4) B. bacilliformis X (4) B. bovis X (4) B. clarridgeiae X(4) B. doshiae X (4) B. elizabethae X (4) B. grahamil X (4) B. henselaeX (4) B. koehlerae X (4) B. quintana X (4) B. schoenbuchensis X (4) B.taylorii X (4) B. tribocorum X (4) B. vinsonii subesp. Arupensis X (4)B. vinsonii subesp. Berkhofii X (4) B. vinsonii subesp. Vinsonii X (4)Coxiella burnetii X (5) Rickettsia aeschlimannii X R. akari X (5) R.australis X (5) R. bellii X (5) R. conori X (5) R. felis X (5) R.Helvetica X (5) R. rickettsii X (5) R. sibirica X R. slovaca X (5) R.prowazekii X R. typhi X (5) Brucella melitensis X (6) Chlamydiapneumoniae X (7) C. psittaci X (7) Escherichia coli X (6) Legionellapneumophila X (8) Leptospira interrogans X (4) Micoplasma pneumoniae X(7) Ochrobactrum antropi X (4) Orientia tsutsugamushi X (5) Pseudomonasaeruginosa X (4) Salmonella enterica Typhi X (4) Streptococcuspneumoniae X (6) Treponema pallidum X (7) Ixodes ricinus X (9)Dermacentor marginatus X (9) Rhipicephalus sanguineus X (9) Apodemussylvaticus X (9) Human DNA  X (10) Internal Control X

Origin of Native DNA:

-   -   1: Positive sample.    -   2: Axenic culture medium, as described in:        -   Benach J L, Coleman J L, and Golightly M G. 1988. A murine            monoclonal antibody binds an antigenic determinant in outer            surface protein A, an immunodominant basic protein of the            Lyme disease spirochete. J. Immunol. 140:265-72.    -   3: Axenic culture medium, as described in:        -   And a P, Segura del Pozo J, Diaz Garcia J M, Escudero R,            Garcia Peña F J, Lopez Velasco M C, Sellek R E, Jimenez            Chillaron M R, Sanchez Serrano L P, Martinez Navarro            J F. 2001. Waterborne outbreak of tularemia associated with            crayfish fishing. Emerg. Infect. Dis. 7 (Suppl):575-82.    -   4: Composition of axenic culture mediums specific to each        species available at: http://cip.pasteur.fr/index.html.en    -   5: Propagation in cellular cultures using the “shell vial”        technique, as described in:        -   Marrero M, Raoult D. 1989. Centrifugation-shell vial            technique for rapid detection of Mediterranean spotted fever            rickettsia in blood culture. Am. J. Trop. Med. Hyg. 40:            197-9.    -   6: “Mueller Hinton” agar culture enriched with 5% of ram blood.    -   7: DNA extracted from slides for indirect commercial        immunofluorescence.    -   8: Axenic culture medium, as described in: Edelstein P H. 1981.        Improved semiselective medium for isolation of Legionella        pneumophila from contaminated clinical and environmental        samples. J. Clin. Microbiol. 14:298-303.    -   9: DNA extracted from pathogen-free specimens.    -   10: DNA extracted from clinical samples of patients with        unrelated diseases.

Synthetic DNA

Synthetic DNA was prepared according to the corresponding sequenceslisted in Tables 1 to 7 (Column 6), by means of consecutive elongationof the DNA chain by PCR, using primers with approximately 70nucleotides, of which approximately 20 nucleotides interoverlapped.

Amplification, Hybridization and Validation

This step included the experimental analysis of the variable regionsdetected earlier using PCR for their validation. The isolated DNA wasamplified using PCR (Saiki et al., (1985) Science 230, 1350; 1354),applying the following temperature cycle table and reaction mixturecomposition, together with the specific primers used previously for saidpurpose.

Temperature Cycles Temperature (° C.) Time Cycles 94 9′ 1 94 15″  60 1′40 65 4′ 65 7′ 1

Reaction mixture composition for a final volume of 50 μL:

-   -   H₂O: According to final DNA volume    -   Buffer Taq Gold LD: 9 μL    -   Cl₂Mg [3 mM]: 6 μL    -   dNTPs [200 mM]: 1 μL×4    -   BSA [0.8 ug/uL]: 4 μL    -   14 specific Primers (SEQ ID 1-2, SEQ ID 7-8, SEQ ID 25-26, SEQ        ID 28-29, SEQ ID 31-32, SEQ ID 36-37, SEQ ID 52-53) [50 pm/μL]:

0.5 μL of each (7 μL)

-   -   Taq Gold LD: 0.5 μL [2.5 units]    -   Problem DNA: maximum 800 ng

The amplicons were sequenced for their validation, verifying that theamplified sequence coincided with the variable sequences inferred frombioinformatic studies. Subsequently, the amplicons were hybridized withspecific probes according to the RLB protocol described by Sjoerd G. T.Rijpkema et al., Journal of Clinical Microbiology, December 1995, p.3091-3095, although applying the following modifications (FIGS. 1 and2A):

-   -   Substrate: Super Signal West Dura (Pierce, Ref: 34075)    -   Probes: used with a concentration of between 0.2 and 3.2        picomoles/microlitre    -   Incubation: at 55° C.    -   Wash: at 52° C.

Hybridization results are shown in FIGS. 1 and 2A, where it is shownthat each of the probes of the invention become joined specifically tothe amplicons of each of the bacterial species detected using the methodof the invention.

Preparation of Samples and Multiple PCR

One of the advantages of using PCR and RLB analysis-based identificationsystems is that pure bacterial cultures are not required. In this mannerand upon validation of the primers and probes using DNA samples of thedifferent species and subspecies listed in Tables 1-6, preparedfollowing the procedures listed in Table 8 and analyzed in duplicate, aMultiple PCR-based analysis of a DNA control mixture prepared underlaboratory conditions was carried out, followed by the RLB test, usingthe specifically designed primers and probes and the previouslyindicated temperature cycles and reaction mixture composition, theresults of which are shown in FIG. 2B. In said figure it is shown thatit was possible to carry out the simultaneous detection of the bacterialspecies present in the sample.

Detection of PCR Inhibitors

An internal amplification control (IAC), which was amplified togetherwith the target DNA, was created for the detection of PCR inhibitors,using specific primers (Table 7) designed according to the conservedregions of the AB183705 sequence (Table 7) belonging to the THC synthasegene of the Cannabis sativa species. Specifically, the IAC ampliconcorresponds to a sequence of 371 pairs of bases, for which a probe wasalso designed (Table 7) for detection during RLB analysis.

Specificity of the Method

The high specificity of this method is based on the specificity of theprimers and their probes, which were tested with another series oforganisms (Table 9), following the method described by the presentinvention, verifying that the formation of amplicons detectable by meansof hybridization (FIG. 3) was not detected in any case.

TABLE 9 Specificity: unrelated species of bacteria, arthropods andmammals used in method development SPECIES RLB RESULT Bacteria 1Brucella melitensis Negative 2 Chlamydia pneumoniae Negative 3 C.psittaci Negative 4 Escherichia coli Negative 5 Legionella pneumophilaNegative 6 Leptospira interrogans Negative 7 Mycoplasma pneumoniaeNegative 8 Ochrobactrum antropi Negative 9 Orientia tsutsugamushiNegative 10 Pseudomonas aeruginosa Negative 11 Salmonella enterica TyphiNegative 12 Streptococcus pneumoniae Negative 13 Treponema pallidumNegative Arthropods Negative 14 Ixodes ricinus Negative 15 Dermacentormarginatus Negative 16 Rhipicephalus sanguineus Negative MammalsNegative 17 Apodemus sylvaticus Negative 18 Human Negative

All references cited and/or discussed in this specification areincorporated herein by reference in their entireties and to the sameextent as if each reference was individually incorporated by reference.

1-15. (canceled)
 16. A kit for detecting bacterial species that causezoonosis, belonging to the genus selected from Anaplasma, Ehrlichia,Bartonella, Borrelia, Coxiella, Francisella, and Rickettsia, comprisingprimers having sequences which comprise or are included within sequencesSEQ ID NOs: 1, 2, 7, 8, 25, 26, 28, 29, 31, 32, 36, and
 37. 17.(canceled)
 18. The kit according to claim 16, wherein it comprises theprimers having the sequences SEQ ID NOs: 1, 2, 7, 8, 25, 26, 28, 29, 31,32, 36 and
 37. 19. The kit according to claim 16, further comprising theprimers SEQ ID NO: 52 and SEQ ID NO:
 53. 20. The kit according to claim16, further comprising probes having sequences which comprise or areincluded within sequences SEQ ID NOs: 3-6, 9-24, 27, 30, 33-35 and38-51.
 21. The kit according to claim 16, further comprising the probeshaving the sequences SEQ ID NOs: 3-6, 9-24, 27, 30, 33-35 and 38-51. 22.The kit according to claim 16, further comprising the probe SEQ ID NO:54.
 23. The kit, according to claim 16, in which the bacterial speciesdetected are: a. Anaplasma phagocytophilum, A. bovis, A. equi, A.marginale, A. centrale and A. ovis. b. Ehrlichia chaffeensis and E.Ewingii; c. Bartonella henselae, B. quintana, B. clarridgeiae, B.elizabethae, B. grahamii, B. vinsonii subspecies berkhofii, B. vinsoniisubspecies vinsonii, B. vinsonii subspecies aurupensis, B.bacilliformis, B. alsatica, B. bovis, B. doshiae, B. koehlerae, B.schoenbuchensis, B. taylori and B. tribocorum; d. All of the speciesbelonging to the genus Borrelia; e. Coxiella burnetii; f. Any subspeciesof Francisella turalensis, including F. tularensis subsp. tularensis, F.tularensis subsp. holarctica and F. tularensis subsp. novicida which arejointly detected, and variation 3523 of the same species and so-calledendosymbionts of different species of ixodides and argasides, which aredetected differentially; and g. The genus Rickettsia, and group thatcauses spotted fever and the group that causes typhus, the speciesRickettsia akari, R. bellii, R. slovaca, R. conorii, R. aeschlimannii,R. ricketsii, R. sibirica, R. helvetica, R. fells, R. australis, R.prowazekii and R. typhy (R. mooserii).
 24. A kit for detecting bacterialspecies that cause zoonosis, belonging to the genus selected fromAnaplasma, Ehrlichia, Bartonella, Borrelia, Coxiella, Francisella andRickettsia, comprising probes having sequences which comprise or areincluded within sequences SEQ ID NOs: 3-6, 9-24, 27, 30, 33-35 and38-51.
 25. The kit according to claim 24, wherein it comprises probeshaving the sequences SEQ ID NOs: 3-6, 9-24, 27, 30, 33-35 and 38-51. 26.The kit according to claim 24, further comprising the probe SEQ ID NO:54.
 27. The kit, according to claim 24, in which the bacterial speciesdetected are: a. Anaplasma phagocytophilum, A. bovis, A. equi, A.marginale, A. centrale and A. ovis; b. Ehrlichia chaffeensis and E.Ewingii; c. Bartonella henselae, B. quintana, B. clarridgeiae, B.elizabethae, B. grahamii, B. vinsonii subspecies berkhofii, B. vinsoniisubspecies vinsonii, B. vinsonii subspecies aurupensis, B.bacilliformis, B. alsatica, B. bovis, B. doshiae, B. koehlerae, B.schoenbuchensis, B. taylori and B. tribocorum; d. All of the speciesbelonging to the genus Borrelia; e. Coxiella burnetii; f. Any subspeciesof Francisella turalensis, including F. tularensis subsp. tularensis, F.tularensis subsp. holarctica and F. tularensis subsp. novicida which arejointly detected, and variation 3523 of the same species and so-calledendosymbionts of different species of ixodides and argasides, which aredetected differentially; and g. The genus Rickettsia, and group thatcauses spotted fever and the group that causes typhus, the speciesRickettsia akari, R. bellii, R. slovaca, R. conorii, R. aeschlimannii,R. ricketsii, R. sibirica, R. helvetica, R. felis, R. australis, R.prowazekii and R. typhy (R. mooserii).